Protoporphyrinogen oxidase is an enzyme catalyzing the end stage reaction of heme synthesis and chlorophyll synthesis, i.e., it catalyzes the reaction that removes six electrons from protoporphyrinogen IX to synthesize protoporphyrin IX. Heme is a cofactor of heme proteins such as hemoglobin and cytochrome, and is an essential molecule for respiration, energy metabolism, and defense against oxygen stress. Heme synthetic pathway commonly exists in microorganisms, plants, and animals, and synthesizes heme from a precursor δ-aminolevulinic acid. Further, in plants, synthetic pathways of heme and chlorophyll share common steps from the precursor δ-aminolevulinic acid until protoporphyrin IX. Protoporphyrinogen oxidase is considered to play a regulatory role in this synthetic pathway. In land plants, the enzyme protoporphyrinogen oxidase, which is responsible for the chlorophyll metabolic system, can be a target enzyme for diphenyl ether (hereinafter referred to as DPE in some cases) herbicides. When DPE herbicides inhibit protoporphyrinogen oxidase activity, protoporphyrinogen IX, a substrate of the enzyme, will accumulate in chloroplast, and eventually the protoporphyrinogen IX will leak out to cytosol where it is oxidized to protoporphyrin IX by peroxidase. When exposed to light and oxygen, protoporphyrin IX may produce singlet oxygen and even other reactive oxygen species. Lipid peroxidation and entailing membrane damage results in rapid death of plant cells (Lee et al., 1993, Plant Physiol., 102, 881). On the other hand, cyanobacteria are known to be able to survive in the presence of DPE herbicide, although the reason or mechanism therefor is not known at all.
Protoporphyrinogen oxidase genes have already been isolated from several organisms. For example, Tobacco PPX1 gene (Genbank accession Y13465) and PPX2 gene (Genbank accession Y13466), Arabidopsis thaliana PPOX gene (Genbank accession D83139), Bacillus subtilis HemY gene (Genbank accession M97208), mouse PPX gene (Genbank accession D45185), human PPX gene (Genbank accession D38537), Saccharomyces cerevisiae PPX gene (Genbank accession Z71381), Escherichia coli hemG gene (Genbank accession X68660) are known.
As an application of protoporphyrinogen oxidases, for example, Japanese Patent Application No. 09-107833 discloses a method to express a protoporphyrinogen oxidase from Bacillus subtilis, which confers resistance against DPE herbicides in a plant and discloses a transgenic plant expressing said protoporphyrinogen oxidase. Further, for example, in Japanese Laid-Open Patent Application No. 09-140381, a protoporphyrinogen oxidase gene of 1.7 kbp length obtainable from Arabidopsis is disclosed as a gene of an enzyme protein in porphyrin biosynthesis system, wherein the gene is suitable for plant cultivation and contains the restriction enzyme EcoR1 recognition nucleotide sequence (5′-GAATTC-3′) at the site 1.3 kbp from its 5′ end. Furthermore, for example, Japanese Patent Application No. 11-346787 discloses a simple method for evaluating inhibitory activity against protoporphyrinogen oxidase derived from rat or Chlamydomonas, said method including the steps of: (1) culturing transformants, which are generated by introducing a DNA fragment composed of operably linked promoter being operable in a host cell and a protoporphyrinogen oxidase gene into a host cell that is deficient in protoporphyrinogen oxidase activity-based growth, and are expressing the protoporphyrinogen oxidase gene present on the DNA fragment, in a medium in the presence or absence of a test compound and measuring the growth rate of the transformants under each condition, wherein the medium does not substantially contain a compound that complements the deficiency in protoporphyrinogen oxidase activity-based growth; and (2) determining the inhibitory activity of the test compound against protoporphyrinogen oxidase activity by determining the degree of inhibition on the transformants' growth via contact with the test compound based on the difference in growth.
On the other hand, in cyanobacteria, a gene analogous to E. coli hemK was speculated to be protoporphyrinogen oxidase from analysis of its gene database. Later, however, said hemK analogous gene of cyanobacteria was proved not to be protoporphyrinogen oxidase in fact. However, proteins homologous to protoporphyrinogen oxidases of other species ever identified have not been found in cyanobacteria gene database, and cyanobacteria protoporphyrinogen oxidase has not been isolated yet (see, e.g., Dmitrii V. Vavilin, Wim F. J. Vermaas, Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria, Physiologia Plantarum, Vol. 115, p. 9, 2002).